The analysis of the structure and functions of proteins has advanced rapidly in recent years as a field of post-genome research. One of the methods that has gained widespread use for the analysis (proteomic analysis) of the structures and functions of proteins uses mass spectrometry to analyze the expression and the primary structure of proteins. The so-called MSn analysis (where n is any integer equal to or greater than 2) which uses quadrupole ion traps, collision induced dissociation (CID) and the like to conduct trapping and fragmenting with regard to specific peaks is proving to be a powerful tool. In general, with the MS2 (=MS/MS) analysis, an ion with a specific mass (more precisely, a mass/charge ratio (m/z)) is selected as a precursor ion from the various ions derived from the specimen to be analyzed. The precursor ion is fragmented using CID, and mass spectrometry is performed on the ions (product ions or fragment ions) that are generated by fragmentation to obtain desired information on the mass or chemical structure of the ions.
To estimate the amino acid sequence of a protein using an MSn analysis such as that described above, the protein is first digested using a suitable enzyme to obtain a peptide fragment mixture. The peptide mixture is then subjected to mass spectrometry. Since the elements that constitute the respective peptides contain stable isotopes of differing masses, peptides having the same amino acid sequence will generate a plurality of peaks of differing masses reflecting the differences in the isotope composition. Those plurality of peaks include a peak of the ion (main ion) that make up only the isotope whose natural abundance ratio is the largest and peaks of ions (isotope ions) that include other isotopes. Those peaks form a group of isotope peaks where the peaks are separated by one Da. intervals when the precursor ion is singly charged.
Next, from the mass spectrum data of a peptide mixture such as described above, one set of isotope peak groups derived from a single peptide is selected as a precursor ion. Mass spectrometry (MS2 analysis) is performed on the ions that are obtained by the fragmentation of the precursor ion. If sufficiently small fragments cannot be obtained by a single fragmentation operation, the fragmentation operation is performed in multiple steps with some MSn by further selecting expected precursor ions.
Either the mass spectrum pattern of the product ions obtained as described above or the mass spectrum pattern of the precursor ion is used as a basis for performing a database search to identify the amino acid sequences with a search engine such as MASCOT, which is provided by Matrix Science Ltd., to determine the amino acid sequence, of the peptide specimen. Alternatively, various analytical software called de novo sequence can be executed on a computer to perform mathematical calculations to estimate the amino acid sequence of the peptide specimen.
Reference 1, a non-patent literature, describes one example of a known prior method for estimating an amino acid sequence using the de novo method. This method uses a mass spectrum based on an MS2 analysis and a mass spectrum based on an MS3 analysis which performs one more step of fragmentation. To briefly explain, the fact that ions that are observed by the MS2 analysis and the MS3 analysis are product ions that have the same terminal (either a C-terminal or a N-terminal) is used to estimate the partial sequences of the peptide. Partial sequences that are determined by a plurality of MS3 analyses are coupled together to estimate the entire amino acid sequence of the peptide. However, the ions that are observed by both the MS2 analysis and the MS3 analysis reflect only some of the product ions that are formed by the fragmentation of the peptide; meaning that that information alone is insufficient for the analysis. One attempt that has been implemented to address this insufficiency is to increase the types of product ion peaks that are collected by using complementary spectrum that are obtained by inverting left and right the mass spectrum (hereinafter simply “MS3 spectrum”) that was obtained with the MS3 analysis. The left and right inversion is performed with respect to the position (mass) of the precursor ion during the MS3 analysis.
Patent Reference 1 describes increasing the product ion peaks by synthesizing a plurality of MSn spectra into a single mass spectrum. However, with this method, not only does the mass spectrum that is synthesized contain much noise but the product ions that are measured using MSn analyses whose value of n is 3 or more contain ions whose bonds are fragmented at two or more locations in the same peptide. This means that as the value of n increases, the synthesized mass spectrum pattern becomes very complicated. The result is that when the amino acid sequence of a peptide is estimated using peaks that appear in the synthesized mass spectrum, the reliability becomes poor despite the time consuming nature of the analysis.
In light of the above-described problems, the present applicant proposed a novel amino acid sequence analysis method in Patent Reference 2. To explain, what is done is to collect the peaks that are commonly present in an MS2 spectrum and an MS3 spectrum, the peaks that are commonly present in the MS2 spectrum and the mass spectrum that is obtained after shifting the MS3 spectrum by the mass of the precursor ion, and the peaks (peaks having complementarity) that are commonly present in the MS2 spectrum and the mass spectrum that is obtained by folding back the MS3 spectrum about the mass of the precursor ion. These many peaks are then categorized and collected as those that belong to the same terminal series, i.e., those that belong to the C-terminal series and the N-terminal series. In this way, many peaks are collected while noise peaks are eliminated. The peak list that is concentrated as b-series and y-series are then provided to an analytical software such as the de novo sequence to estimate the amino acid sequence. This improves the accuracy of the estimation of the amino acid sequence of a peptide.
However, even with this method, depending on the type of peptide that is analyzed, the reliability of the data that is provided to the analytical software decreases and the accuracy of the amino acid sequence estimation drops. There are a number of reasons for this including the fact that, even with the MSn spectrum where the number of fragmentation operation steps is increased, the ions that correspond to the peaks where mass commonality or complementarity is not found are not used and the fact that errors may be made in the classification into C-terminal or N-terminal of ions that are categorized based on mass commonality or complementarity that is found.    Patent Reference 1: U.S. Pat. No. 6,624,4087 Specification    Patent Reference 2: JP Unexamined Patent Application Publication 2007-278712A    Non-Patent Reference 1: Z. Zhang and one other, “De Novo Peptide Sequencing by Two-Dimensional Fragment Correlation Mass Spectrometry,” Analytical Chemistry, Vol. 72, No. 11, Jun. 1, 2000, pp. 2337-235